Enzyme-labeled antibody technology

Enzyme markers include enzyme-labeled antigens, enzyme-labeled antibodies and enzyme-labeled SPA. The quality of enzyme markers is directly related to the success of immunoenzyme technology, so it is called a key reagent. The most commonly used enzyme labels are enzyme-labeled antibodies, which are formed by linking enzymes and specific antibodies through appropriate methods. The quality of enzyme-labeled antibodies mainly depends on the enzymes and antibodies with good purity, strong activity and high affinity, followed by a good preparation method. At present, high-quality enzymes (such as horseradish peroxidase, HRP for short) are already available in China. High-quality antibodies can be obtained by extraction and purification. In the preparation method, the method with high yield, which does not affect the activity of the conjugate and does not mix interfering substances, and is easy to operate is suitable.

1. Selection of working concentration

In immunoenzyme technology, the first factor to be determined is the working concentration of the enzyme marker. Because small changes in the concentration of enzyme markers can cause large fluctuations in the test results. In addition, because the concentration is too high, non-specific reactions can be increased, while the concentration is too low can affect the sensitivity of the measurement. Therefore, the working concentration must be accurately titrated before the formal test.

The titration method of enzyme-labeled antibody is: physical adsorption of the antigen (or antibody) on the solid phase carrier, and then a series of diluted enzyme-labeled antibody (or anti-Ig antibody) and the antigen (or antibody) adsorbed on the carrier The reaction determines the titer of the enzyme-labeled antibody based on the degree of color reaction between the enzyme and the substrate, or the working concentration.

The steps are: first dilute the antigen (or antibody) with 0.05M PH9.6 coating buffer to about 10μg / ml, add 0.1ml to the polystyrene plate well, overnight at 4 ℃, and wash with the washing buffer the next day 3 times. Enzyme-labeled antibody was diluted with 1% BSA-PBS solution to 1: 100, 1: 200, 1: 400, 1: 800, 1: 1600 ... (depending on the antibody titer), and added to the reaction wells respectively , Two wells per dilution, 0.1ml per well, washed after incubating at 37 ° C for 1 hour. Then add substrate solution, 0.1ml per well, 37 ℃ for 10-30 minutes. The reaction was stopped with 2M H2SO4 0.05ml.

As a result, the OD value of each well was mainly read by ELISA colorimeter. The titration curve is drawn with OD as the vertical coordinate and the concentration of the conjugate as the horizontal coordinate. According to the curve, the dilution of the enzyme-labeled antibody when the OD value is about 1.0 and the slope of the curve is maximum is the working concentration of the marker.

See the ELISA section for reagents and equipment for the test.

It should be noted that this method is a direct ELISA method, and the measured working concentration may differ from the optimal concentration in practical application by several titers. This requires the establishment of an ELISA experimental system, so the actual working concentration should be further determined (square array method can be used) to achieve the most suitable experimental conditions.

2. Enzyme preparation and its substrate

Any enzyme that is non-toxic and can exhibit a colored chemical reaction can be used as a label in principle. However, the enzyme used as a labeled antibody should meet the following requirements: (1) convenient source and easy purification; (2) high specific activity and stable properties; (3) enzyme activity and amount can be determined by a simple method. The enzymes currently commonly used in immunoenzyme technology are horseradish peroxidase (HRP) and alkaline phosphatase (AP), followed by glucose oxidase, β-galactosidase, lysozyme and malate dehydrogenase Wait.

Horseradish Peroxidase (HRP) is the most commonly used because of its high specific activity, stability, small molecular weight, and easy preparation of pure enzymes. HRP is widely distributed in the plant kingdom, and is high in horseradish. It is a glycoprotein composed of colorless enzyme protein and brown iron porphyrin, with a sugar content of 18%. HRP is composed of multiple isozymes with a molecular weight of 40,000 and an isoelectric point of PH3-9. The optimal pH catalyzed by the enzyme is slightly different due to different hydrogen donors, but most of them are around PH5. The enzyme is soluble in water and ammonium sulfate solution below 58% saturation. The maximum absorption spectra of the prosthetic group of HRP and the enzyme protein are 403nm and 275nm, respectively, and the purity of the enzyme is generally expressed by the ratio OD403nm / OD275nm RZ (German Reinheit Zahl). High-purity enzyme RZ value should be around 3.0 (up to 3.4). The smaller the RZ value, the more non-enzymatic protein. It is worth noting that purity does not indicate enzyme activity. For example, when the enzyme is denatured, the RZ value can remain unchanged.

The HRP catalytic reaction requires substrates hydrogen peroxide (H2O2) and hydrogen donor (DH2). The hydrogen donors are mostly colorless vat dyes, and colored oxidative dyes (D) can be generated through the reaction. The process of the enzymatic reaction is as follows:

HRP

DH2 + H2O2──── → D + 2H2O

There are many types of hydrogen donors, and the characteristics of the products formed are different. For example, the reaction product of DAB (3.3-diaminobenzidine) is an insoluble precipitate and has an electron density, so it is suitable for immunoenzyme staining or electron microscopic observation. 5AS (5-aminosalicylic acid) was used in early ELISA, but its solubility is not large enough, and the blank hole is not easy to control to colorless, and it has been rarely used. The characteristic of OT (o-tolidine) is that it can produce bright blue-green products with high sensitivity, but it is greatly affected by temperature in the reaction, and because the products are unstable, it needs to be measured in a short time. Currently widely used and satisfactory hydrogen donors are: OPD (o-phenylenediamine) and TMB (tetramethylbenzidine). The product formed by the former is deep orange or brown, and the product of the latter is blue-green. Both have good solubility. The color is stable in dark places. The blank can be nearly colorless. The latter is reported to be higher than the former in sensitivity. 4 More than times. In addition, there is another kind of hydrogen donor called ABTS [2, 2-side-nitro-bis (3-ethylbenzothipyrroline-6sulfonic acid)], the reaction product is blue-green, and the sensitivity and stability are both it is good. Especially in terms of carcinogenic potential, both ABTS and TMB are worthy hydrogen donors.

Since H2O2, the substrate of HRP, is itself an enzyme inhibitor, the amount of H2O2 used in the enzymatic reaction cannot be excessive. It should be controlled to reach a peak after a short period of time after reaction (indicating that H2O2 has been exhausted). In this way, even if the time is extended, the color of the reaction product will not be increased.

3. The method of HRP labeling antibody

There are many methods for cross-linking enzymes and antibodies, and different methods can be used depending on the structure of the enzyme. For the preparation of HRP conjugates, glutaraldehyde two-step method and sodium periodate method can be used. In particular, the simple sodium periodate method is more commonly used.

Folder91E3.gif (1087 bytes) Glutaraldehyde two-step method

1. Principle: Glutaraldehyde is a bifunctional reagent, through which the aldehyde group is covalently bound to the enzyme and the amino group on the immunoglobulin to form the enzyme-glutaraldehyde-immunoglobulin conjugate.

2. Marking steps:

(1) Weigh 25mg of HRP and dissolve it in 1.25% glutaraldehyde solution and let stand at room temperature overnight.

(2) The enzyme solution after the reaction was passed through a Sephadex G-25 chromatography column and eluted with physiological saline. The flow rate was controlled at 1 ml / 1 minute, and the brown effluent was collected. If the volume is greater than 5ml, concentrate with PEG to 5ml. Place in a 25ml small beaker and stir slowly.

(3) Dilute 12.5 mg of the antibody to be labeled with physiological saline to 5 ml, and drop it into the enzyme solution dropwise with stirring.

(4) Use 0.25ml of 1M PH9.5 carbonate buffer and continue stirring for 3? Hours.

(5) Add 0.25ml of 0.2M lysine. After mixing, let stand at room temperature for 2 hours.

(6) Add an equal volume of saturated ammonium sulfate dropwise with stirring, and set at 4 ° C for 1 hour.

(7) Centrifuge at 3000 rpm for half an hour and discard the supernatant. The precipitate was washed twice with half-saturated ammonium sulfate, and finally the precipitate was dissolved in a small amount of 0.15M PH7.4 PBS.

(8) Put the above solution into a dialysis bag, dialyze against 0.15M PH7.4 PB buffered saline, remove the ammonium ion (detected with naphthalene reagent), centrifuge at 10,000rpm for 30 minutes to remove the precipitate, the supernatant is the enzyme The conjugate is stored frozen after aliquoting.

3. Result judgment:

(1) Qualitative and titer titration: use specific antigen (or antibody) and enzyme labeled antibody (or anti-immunoglobulin antibody) for two-way agar diffusion test or immunoelectrophoresis test. The precipitation arc is then developed with the substrate of the enzyme, and its activity can be initially identified. Finally, the enzyme conjugate is titrated by direct ELISA (or in a formal experimental system) (see the selection of the working concentration in this section (3)).

(2) Quantitative and molar ratio measurement: it can be measured with a spectrophotometer (optical path 1cm).

Enzyme amount (mg / ml) = OD403nm × 0.4

IgG amount (mg / ml) = OD280nm-OD403nm × 0.42) × 0.94 × 0.62

Enzyme amount (mg / ml) IgG amount (mg / ml) Enzyme amount molar ratio = ──────── ÷ ─────── = ─── × 4
40,000 160,000 IgG amount

(3) The labeling procedure of this method is relatively simple and repeatable. The disadvantage is that the enzyme utilization rate is low, generally only 2 to 4% of the enzyme is combined with the protein.

4. Reagents and equipment:

(1) 0.1M PH6.8 phosphate buffered saline (PBS): take 0.2M Na2HPO> 4 49ml, 0.2M NaH2PO> 4 51ml, 1.8g NaCl, and add distilled water to 200ml.

(2) 1.25% glutaraldehyde solution: mix 50ml of 25% glutaraldehyde with 1ml of PBS at pH 6.8.

(3) 1M PH9.5 carbonate buffer: Mix 3ml of 1M sodium carbonate with 7ml of 1M sodium bicarbonate.

(4) 0.2M lysine solution: Weigh 29.2mg of lysine dissolved in 1ml of 0.01M PH9.5 carbonate buffer.

(5) 0.15M PH7.4 PBS and physiological saline.

(6) PH7.8 saturated ammonium sulfate solution and semi-saturated ammonium sulfate solution.

(7) Naphthalene reagent and polyethylene glycol (PEG, MW2000).

(8) Purified specific antibody or anti-Ig antibody.

(9) HRP (RZ> 3.0).

(10) Sephadex G-25 chromatography column (2cm × 50cm).

(11) Stirrer, spectrophotometer, centrifuge.

(12) Dialysis bags, large and small beakers, test tubes, straws, etc.

Simple sodium periodate method

In this method, NaIO4 first oxidizes the sugar molecules on the surface of HRP into aldehyde groups, and then combines with the amino group on Ig. The yield of the enzyme-labeled antibody obtained is high. Combined with the enzyme, there is no significant loss of the activity of the enzyme and Ig, which is currently the most commonly used method.

1. Principle: In the classic sodium periodate method, dinitrofluorobenzene needs to be used to block the remaining α- and ε amino groups on the HRP to avoid cross-linking between enzyme molecules. Later, Wilson et al. Used NaIO4 to oxidize HRP at low pH, thereby eliminating the dinitrofluorobenzene blocking HRP step. The hydroformylase formed by the oxidation of HRP by NaIO4 can be linked to the amino group of the antibody molecule to form stromalline, which can be further reduced with NaBH4 (or ethanolamine) to generate stable enzyme-labeled antibodies.

2. Marking steps:

(1) Weigh 5mg HRP and dissolve it in 1ml distilled water.

(2) Add 0.2ml of freshly prepared 0.1M NaIO4 solution to the upper solution and stir at room temperature in the dark for 20 minutes.

(3) Put the above solution into a dialysis bag, dialyze against 1mM sodium hydroxide buffer with pH 4.4, and overnight at 4 ℃.

(4) Add 20μl of 0.2M PH9.5 carbonate buffer to raise the pH of the above-mentioned aldehyde RP to 9.0 ~ 9.5, then immediately add 10mg IgG (antibody, or SPA5mg) in 1ml 0.01M carbonate buffer In the liquid, stir gently at room temperature in the dark for 2 hours.

(5) Add 0.1ml of newly prepared 4mg / ml NaBH4 solution, mix well, and set at 4 ℃ for 2 hours.

(6) Put the above solution into a dialysis bag, dialyze against 0.15M PH7.4 PBS, overnight at 4 ℃.

The remaining steps (purification) are the same as (6), (7) and (8) of the glutaraldehyde labeling step.

3. Result judgment:

Except for the calculation of the amount of the marker IgG, which is slightly different, the rest are the same as the glutaraldehyde method.

IgG amount (mg / ml) = (OD280nm-OD403nm × 0.3) × 0.62

4. Reagents and equipment:

(1) 0.1M NaIO4: Weigh 241mg sodium periodate (Guangzhou Chemical Reagent Factory, batch number 830602) and dissolve it in 10ml of distilled water.

(2) 1mM PH4.4 sodium acetate buffer:

0.2M NaAc (1.361g / 50ml) 3.7ml

0.2M HAc (0.601ml / 50ml) 6.3ml

Add distilled water to 2,000ml.

(3) 0.2M PH9.5 carbonate buffer:

Na2CO3 0.32g

NaHCO3 0.586g

Add distilled water to 50ml

Then make a 20-fold dilution with distilled water to make 0.01M PH9.5 carbonate buffer.

(4) NaBH4 solution (4mg / ml):

Before use, weigh out NaBH44mg and dissolve it in 1ml of distilled water.

(5) For other reagents and equipment, please refer to the glutaraldehyde labeling method.

Four, matters needing attention

1. Under the condition of high-quality HRP, the antibody to be labeled should also have high activity, high titer (minimum 1:16), high purity and good affinity. This is the first thing to ensure that the label has high titer and good immune activity. condition.

2. The PH, concentration and dosage of the reagents used must be strictly controlled. The reagents used are best (or necessary) freshly prepared. The glutaraldehyde used in the glutaraldehyde labeling method should be fresh and pure, because glutaraldehyde can be stored for a long time to form a condensate (impurities). Otherwise, the mark effect will be affected.

3. Keep away from light when stirring at room temperature. The indoor temperature is generally 25 ℃. Before each dialysis, the marker must be carefully checked to prevent leakage.

4. Concentrated markers are quite stable. 30-30% glycerin is often added and stored at -10 ℃. It can be stored for 1 to 2 years at 4 ℃, but diluted to 1:10, it can only be stored for several weeks. The prepared use solution should be used up within 12 hours. Avoid repeated freezing and thawing.

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